Project Summary. Neuroimmune Sensitization and Circuit Pathogenesis: An Alzheimer's supplement to P60-AA011605 in response to (PA-18-591). Emerging data suggest that Toll-like receptors (TLRs) and neuroimmune signaling are involved in Alzheimer's disease (AD). Neuroimmune signaling and ethanol drinking induce neuroimmune and microglial activation. Additionally, microglial priming of proinflammatory responses increases over time. Our studies find that ethanol and stress increase brain microglial, astrocyte and neuronal neuroimmune gene expression with progressive increases over time. Microglia are unique brain neuroimmune cells that are sensitized to proinflammatory responses by environmental and genetic factors that induce Toll-like receptors. We discovered that cycles of ethanol exposure, similar to stress cycles, lead to long-lasting changes in expression of neuroimmune genes, including neuronal Toll-like receptor 3 (TLR3). We have found that TLR3 expression is increased in post-mortem human alcoholic brains, and is increased by ethanol exposure in both cortex and striatum in rats. These are highly relevant findings given that TLR3 expression, both protein immunoreactivity and gene mRNA expression, are also increased in post-mortem Alzheimer's disease (AD) brain tissue. Neuroimmune gene induction of oxidative stress and neurodegeneration are well known to contribute to AD. Importantly, our TLR3 finding is consistent with increases in TLR3 signaling persisting for long periods that could sensitize and contribute to neurodegeneration and the development of AD. This result emphasizes TLR3 as a potential therapeutic target for AD. Additionally, we find that treatment with TLR3 agonist polyinosinic:polycytidylic acid (poly(I:C)) results in escalated neuroimmune gene induction and markers of activated astrocytes and neuronal synapse markers. Moreover, we observe parallel poly(I:C)-induced increases in TLR3 gene expression, Gi-coupled Group II metabotropic glutamate receptors (mGluR2 and 3) consistent with increased glutamate signaling. We propose to expand our analyses to include AD markers (APP, Tau, beta-secretase [BACE1], GSK3?, A?42), and mRNA determination of TLR3 and other TLR signaling genes (MyD88, IRF, STAT, NFkB), as well as neuroimmune signaling genes (TLR3, HMGB1, MCP1, IL6, TNF?). We propose to extend our studies of glutamatergic synapse markers (PSD95, mGluR2, mGluR3, GLT1, AMPA, NMDA subunits) to include the brain regions involved in AD pathology (prefrontal cortex, entorhinal cortex and hippocampus). In addition, we propose to assess microglial markers (Iba1, CD11b), as well as microglia synaptic regulatory proteins (C1q, CR3, CD200, CD200R), astrocyte markers (Aq4, GFAP, GLT1, ALDH), and astrocyte synaptic proteins (i.e., thrombospondins). In this supplement, we will expand our investigation of TLR3 neuroimmune signaling synaptic changes to test the hypothesis that poly(I:C)-TLR3 activation induces neuroadaptations in glutamatergic synapses and glia that contribute to AD pathology. As such, experiments in Aim 1 will examine changes in neuroimmune signaling and markers of AD-related pathology. Experiments in Aim 2 will focus on neuroadaptations in glutamatergic synapses and glia that may contribute to AD pathology. Together these studies will determine the impact of TLR3 sensitization on markers of AD pathology across multiple brain regions and brain cell types for neuroimmune, synaptic and AD degeneration genes.